Genes that are modulated by posttranscriptional gene silencing

Abstract

The invention provides a method to identify genes that are modulated by posttranscriptional gene silencing as well as regulatory elements and methods to modulate gene expression by posttranscriptional gene silencing.

What is claimed is:  
     
         1 . An isolated polynucleotide comprising a plant nucleotide sequence 
 (a) selected from the group consisting of SEQ ID NOs: 1 to 341 or a fragment thereof which encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide;    (b) having substantial similarity to (a);    (c) having at least 15 nucleotides and capable of hybridizing to (a) or the complement thereof under stringent conditions;    (d) having at least 15 nucleotides and capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in SEQ ID NOs: 1 to 341 or the complement thereof under stringent conditions;    (e) complementary to (a), (b) or (c); or    (f) which is a reverse complement of (a), (b) or (c).    
     
     
         2 . An isolated polynucleotide comprising a nucleotide sequence having at least 70% nucleic acid sequence identity with the polynucleotide of  claim 1  or the complement thereof.  
     
     
         3 . An isolated polynucleotide comprising a nucleic acid sequence having at least 80% nucleic acid sequence identity with the polynucleotide of  claim 1  or the complement thereof.  
     
     
         4 . An isolated polynucleotide comprising a nucleic acid sequence having at least 90% nucleic acid sequence identity with the polynucleotide of  claim 1  or the complement thereof.  
     
     
         5 . An isolated polynucleotide comprising a nucleic acid sequence having at least 98% nucleic acid sequence identity with the polynucleotide of  claim 1  or the complement thereof.  
     
     
         6 . The polynucleotide of  claim 1 , wherein the polynucleotide comprises a plant nucleotide sequence encoding a polypeptide which modulates gene expression through posttranscriptional gene silencing.  
     
     
         7 . The polynucleotide of  claim 6 , wherein the polynucleotide comprises a plant nucleotide sequence encoding a polypeptide that is substantially similar to a polypeptide with a amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 342, or a fragment thereof which has substantially the same activity as the full-length polypeptide.  
     
     
         8 . A vector comprising the polynucleotide of  claim 1 .  
     
     
         9 . An expression cassette comprising the polynucleotide of  claim 1 , operably linked to a regulatory sequence.  
     
     
         10 . The expression cassette of  claim 9 , wherein the regulatory sequence is selected from the group consisting of a promoter, an operator, an enhancer, a repressor binding site and a transcription factor binding site.  
     
     
         11 . The expression cassette of  claim 9 , wherein the polynucleic acid segment is oriented relative to the promoter such that an antisense message is transcribed.  
     
     
         12 . A cell comprising the expression cassette of  claim 9 .  
     
     
         13 . The cell of  claim 12 , wherein the cell is a plant cell.  
     
     
         14 . The cell of  claim 12 , wherein the cell is a monocotyledonous cell, a dicotyledonous cell, a cereal plant cell, a Rosidea cell, a Brassicales cell, an Arabidopsis cell, a rice plant cell, a wheat plant cell, a barley plant cell or a maize plant cell.  
     
     
         15 . A recombinant cell, wherein the genome of the cell is augmented with the expression cassette of  claim 9 .  
     
     
         16 . The recombinant cell of  claim 15 , wherein the cell is a plant cell.  
     
     
         17 . A mutagenesis cassette comprising an intervening nucleic acid sequence linked on both ends to a flanking nucleic acid sequence that hybridizes under low stringency conditions to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 341, or a complement thereof.  
     
     
         18 . The mutagenesis cassette of  claim 17 , wherein the intervening nucleic acid sequence is a selectable marker.  
     
     
         19 . A vector comprising the mutagenesis cassette of  claim 17 .  
     
     
         20 . A polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence having at least 70% sequence identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 341 or a complement thereof.  
     
     
         21 . A polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence having at least 80% sequence identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 341, or a complement thereof.  
     
     
         22 . A polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence having at least 90% sequence identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 341, or a complement thereof.  
     
     
         23 . A polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence having at least 98% sequence identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 341, or a complement thereof.  
     
     
         24 . The polypeptide of  claim 20 , wherein the polypeptide modulates gene expression by posttranscriptional gene silencing.  
     
     
         25 . A method to modulate gene expression within a cell by posttranscriptional gene silencing comprising: 
 a) introducing into a cell a polynucleotide having at least 70% nucleic acid sequence identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 341, or a complement thereof, operably linked to a regulatory sequence; and    (b) expressing the polynucleotide in the cell to form a product, wherein the product modulates gene expression within the cell.    
     
     
         26 . The method of  claim 25 , wherein the cell is a monocotyledonous cell, a dicotyledonous cell, a cereal plant cell, a Rosidea cell, a Brassicales cell, an Arabidopsis cell, a rice plant cell, a wheat plant cell, a barley plant cell or a maize plant cell.  
     
     
         27 . The method of  claim 25 , wherein the regulatory sequence is selected from the group consisting of a promoter, an operator, an enhancer, a repressor binding site and a transcription factor binding site.  
     
     
         28 . A method for isolating a polynucleic acid fragment containing a regulatory element that modulates expression of a polynucleic acid segment within a cell by posttranscriptional gene silencing, comprising: 
 a) hybridizing an oligonucleotide probe, wherein said oligonucleotide probe hybridizes to a polynucleic acid segment having at least 70% nucleic acid sequence identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 341 and having expression that is modulated within a cell by posttranscriptional gene silencing, to a polynucleic acid fragment that corresponds to genomic DNA obtained from the cell to form a complex, wherein the polynucleic acid fragment comprises the regulatory element and the polynucleic acid segment; and    b) isolating the polynucleic acid fragment from the complex formed by hybridization of the oligonucleotide to the polynucleic acid fragment containing the regulatory element.    
     
     
         29 . The method of  claim 28 , further comprising sequencing the isolated polynucleic acid fragment.  
     
     
         30 . The method of  claim 28 , wherein the oligonucleotide probe hybridizes under low stringency conditions to a polynucleic acid segment selected from the group consisting of SEQ ID NOs: 1 to 341, or a complement thereof.  
     
     
         31 . The method of  claim 28 , wherein the cell is a plant cell.  
     
     
         32 . A method for isolating the regulatory element from the polynucleic acid fragment of  claim 28 , comprising: 
 a) screening for expression of a detectable marker in cells transformed with a vector that encodes the detectable marker and contains a portion of the polynucleic acid fragment, wherein the portion of the polynucleic acid fragment causes expression of the detectable marker to be modulated by posttranscriptional gene silencing; and    b) isolating the regulatory element from the vector obtained from the cell wherein expression of the detectable marker is modulated by posttranscriptional gene silencing.    
     
     
         33 . A regulatory element isolated according to the method of  claim 32 .  
     
     
         34 . A method for obtaining an amplification product containing a regulatory element that modulates expression of a polynucleic acid segment in a cell by posttranscriptional gene silencing, comprising: 
 a) amplifying a nucleic acid sequence that contains the regulatory element by hybridizing a first oligonucleotide primer to a polynucleic acid fragment comprising the polynucleic acid segment and the regulatory element, and hybridizing a second degenerate oligonucleotide primer to the polynucleic acid fragment at a position that is 5′ or 3′ to an open reading frame containing the polynucleic acid segment, and amplifying the nucleic acid sequence between the first primer and the second primer by polymerase chain reaction to form an amplification product, wherein the polynucleic acid fragment is genomic DNA from the cell; and    b) isolating the amplification product formed by the polymerase chain reaction between the first and the second primer to obtain the regulatory element.    
     
     
         35 . The method of  claim 34 , further comprising sequencing the amplification product.  
     
     
         36 . The method of  claim 34 , wherein the first oligonucleotide primer hybridizes to a nucleic acid segment selected from the group consisting of SEQ ID NOs: 1 to 341, or a complement thereof.  
     
     
         37 . An amplification product isolated according to the method of  claim 34 .  
     
     
         38 . A method for isolating the regulatory element of  claim 34  from the amplification product comprising: 
 a) screening for expression of a detectable marker in cells that were transformed with a vector that encodes the detectable marker and contains a portion of the amplification product, wherein the portion of the amplification product causes expression of the detectable marker to be modulated by posttranscriptional gene silencing; and  
 b) isolating the regulatory element from the vector obtained from the cell wherein expression of the detectable marker is modulated by posttranscriptional gene silencing.  
 
     
     
         39 . A regulatory element isolated according to the method of  claim 38 .  
     
     
         40 . An expression cassette comprising the regulatory element of  claim 33  operably linked to an open reading frame.  
     
     
         41 . An expression cassette comprising the regulatory element of  claim 39  operably linked to an open reading frame.  
     
     
         42 . A method to augment a plant genome comprising: 
 a) contacting a plant cell with the polynucleotide of  claim 1  to produce a transformed plant cell; and    b) growing the transformed plant cell to produce a differentiated transformed plant.    
     
     
         43 . The method of  claim 42 , wherein the plant cell is selected from the group consisting of a monocotyledonous cell, a dicotyledonous cell, a cereal plant cell, a wheat plant cell, a rice plant cell, a barley cell, a maize plant cell, a Rosidea cell, a Brassicales cell, and an Arabidopsis cell.  
     
     
         44 . A transgenic plant comprising a polynucleotide of  claim 1 .  
     
     
         45 . A transgenic plant comprising the polynucleotide of  claim 2 .  
     
     
         46 . The plant of  claim 45 , wherein the plant is a monocotyledonous plant, a dicotyledonous plant, a cereal plant, a wheat plant, a rice plant, a barley plant, a maize plant, a Rosidea plant, a Brassicales plant or an Arabidopsis plant.  
     
     
         47 . A product produced by a plant of  claim 45 , wherein the product is a seed, a fruit, a vegetable, a transgenic plant, a progeny plant or products of the progeny plant.  
     
     
         48 . A method to create a mutant cell comprising: contacting a cell with the mutagenesis cassette of  claim 18  to produce a cell having a deletion in a gene that is regulated by posttranscriptional gene silencing.  
     
     
         49 . The method of  claim 48 , wherein the cell is a plant cell.  
     
     
         50 . A method for identifying a first polypeptide that forms a complex with a second polypeptide within a cell, comprising: 
 a) expressing within the cell the second polypeptide, wherein said second polypeptide is a fusion polypeptide that comprises a marker polypeptide and a polypeptide having expression that is modulated by posttranscriptional gene silencing, so that a complex is formed that contains the first polypeptide and the second polypeptide; and    b) separating the complex by using the marker polypeptide and identifying the first polypeptide contained within the complex.    
     
     
         51 . The method of  claim 50 , wherein the second polypeptide is encoded by a polynucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 341, or a complement thereof.  
     
     
         52 . The method of  claim 50 , wherein the marker polypeptide is glutathione-S-transferase.  
     
     
         53 . The method of  claim 50 , wherein the marker polypeptide is an epitope for an antibody.  
     
     
         54 . A method for determining a polynucleotide that encodes a first polypeptide that interacts with a second polypeptide whose expression is modulated by posttranscriptional gene silencing, comprising: 
 a) mating a first haploid yeast cell, wherein said first haploid yeast cell contains a construct comprising a polynucleotide that encodes the first polypeptide fused to a DNA-binding domain or to an RNA polymerase activation domain, with a second haploid yeast cell, which contains a construct comprising a nucleic acid sequence selected from SEQ ID NOs: 1 to 341 fused to a DNA-binding domain or an RNA polymerase activation domain, wherein the first haploid yeast strain and the second haploid yeast strain do not contain a construct that encodes for the same DNA-binding domain or RNA-polymerase activation domain and either of the first or the second haploid yeast strain comprises a two-hybrid reporter gene; and    b) determining the sequence that encodes the first polypeptide by sequencing the construct that expresses the first polypeptide as indicated by measuring the activation of the two-hybrid reporter gene in the diploid yeast strain.    
     
     
         55 . An isolated polynucleotide segment that hybridizes under low stringency conditions to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 341, or a complement thereof.  
     
     
         56 . An isolated polynucleotide segment that encodes a polypeptide having substantial similarity to a polypeptide encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 341, or a complement thereof.  
     
     
         57 . An isolated polynucleotide segment having a similarity value less than 1×10 −5  as determined by BLAST search using default parameters to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 341, or a complement thereof.  
     
     
         58 . An isolated polynucleotide segment having a similarity value less than 1×10 −10  as determined by BLAST search using default parameters to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 341, or a complement thereof.  
     
     
         59 . An isolated polynucleotide segment having a similarity value less than 1×10 −20  as determined by BLAST search using default parameters to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 341, or a complement thereof.  
     
     
         60 . An isolated polynucleotide segment having a similarity value less than 1×10 −25  as determined by BLAST search using default parameters to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 341, or a complement thereof.  
     
     
         61 . A cell comprising the isolated polynucleotide segment of  claim 57 .  
     
     
         62 . A transgenic plant comprising the polynucleic acid segment of  claim 57 .  
     
     
         63 . A computer-readable medium having stored thereon a data structure comprising: 
 a) at least one nucleic acid sequence that encodes a polypeptide which confers post-transcriptional gene silencing onto a plant and has at least 70% nucleic acid sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 341, or a complement thereof; and    b) a module receiving the nucleic acid sequence which compares the nucleic acid sequence to another nucleic acid sequence.    
     
     
         64 . The computer readable medium of  claim 63 , wherein the medium is selected from the group consisting of magnetic tape, optical disk, CD-ROM, random access memory, volatile memory, non-volatile memory and bubble memory.  
     
     
         65 . A computer-readable medium having stored thereon computer executable instructions for performing a method comprising: 
 a) receiving at least one nucleic acid sequence having at least 70% nucleic acid sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 341, 473 to 539; 540 to 673, and 674 to 779, or a complement thereof; and    b) comparing the nucleic acid sequence to another nucleic acid sequence.    
     
     
         66 . The computer readable medium of  claim 65 , wherein the medium is selected from the group consisting of magnetic tape, optical disk, CD-ROM, random access memory, volatile memory, non-volatile memory and bubble memory.  
     
     
         67 . A method of determining the silencing status of a plant comprising 
 (a) obtaining an RNA expression profile for a subset of genes comprising one or more genes the expression of which is modulated by posttranscriptional gene silencing (PTGS); and    (b) comparing the expression profile obtained with the profile of a plant of the same species that does not have posttranscriptional gene silencing.